Eukaryotic cells constantly degrade and replace cellular protein. This permits the cell to selectively and rapidly remove proteins and peptides having abnormal conformations, to exert control over metabolic pathways by adjusting levels of regulatory peptides, and to provide amino acids for energy when necessary, as in starvation. See Goldberg, A. L. & St. John, A. C. Annu. Rev. Biochem. 45:747-803 (1976). The cellular mechanisms of mammals allow for multiple pathways for protein breakdown. Some of these pathways appear to require energy input in the form of adenosine triphosphate ("ATP" ). See Goldberg, A. L. & St. John, supra.
Multicatalytic protease (MCP, also referred to as "multicatalytic proteinase," "proteasome," "multicatalytic proteinase complex," "multicatalytic endopeptidase complex," "20S proteasome" and "ingensin") is a large molecular weight (700 kD) eukaryotic cytoplasmic proteinase complex which plays a role in at least two cellular pathways for the breakdown of protein to peptides and amino acids. See Orlowski, M. Biochemistry 29(45) 10289-10297 (1990). The complex has at least three different types of hydrolytic activities: (1) a trypsin-like activity wherein peptide bonds are cleaved at the carboxyl side of basic amino acids; (2) a chymotrypsin-like activity wherein peptide bonds are cleaved at the carboxyl side of hydrophobic amino acids; and (3) an activity wherein peptide bonds are cleaved at the carboxyl side of glutamic acid. See Rivett, A. J. J. Biol. Chem. 264:21 12215-12219 (1989) and Orlowski, supra.
One route of protein hydrolysis which involves MCP also involves the polypeptide "ubiquitin." Hershko, A. & Ciechanover A. Annu. Rev. Biochem. 51:335-364 (1982). This route, which requires MCP, ATP and ubiquitin, appears responsible for the degradation of highly abnormal proteins, certain short-lived normal proteins and the bulk of proteins in growing fibroblasts and maturing reticuloytes. See Driscoll, J. and Goldberg, A. L. PNAS 86:787-791 (1989). Proteins to be degraded by this pathway are covalently bound to ubiquitin via their lysine amino groups in an ATP-dependent manner. The ubiquitin-conjugated proteins are then degraded to small peptides by an ATP-dependent protease complex by the 26S proteasome, which contains MCP as its proteolytic core. Goldberg, A. L. & Rock, K. L. Nature 357:375-379 (1992).
A second route of protein degradation which requires MCP and ATP, but which does not require ubiquitin, has also been described. See Driscoll, J. & Goldberg, A. L., supra. In this process, MCP hydrolyzes proteins in an ATP-dependent manner. See Goldberg, A. L. & Rock, K. L., supra. This process has been observed in skeletal muscle. See Driscoll & Goldberg, supra. However, it has been suggested that in muscle, MCP functions synergistically with another protease, multipain, thus resulting in an accelerated breakdown of muscle protein. See Goldberg & Rock, supra.
The relative activities of cellular protein synthetic and degradative pathways determine whether protein is accumulated or lost. The abnormal loss of protein mass is associated with several disease states such as muscular dystrophy, cardiac cachexia, emphysema, leprosy, malnutrition, osteomalacia, child acute leukemia, and cancer cachexia. Loss of muscle mass is also observed in aging, long term hospitalization or long term confinement to bed, and in chronic lower back pain.
With denervation or disuse, skeletal muscles undergo rapid atrophy which leads to a profound decrease in size, protein content and contractile strength. This atrophy is an important component of many neuromuscular diseases in humans. Enhancement of protein breakdown has been implicated as the primary cause of muscle wasting in denervation atrophy, See Furono, K. et al. J. Biochem. 265/15:8550-8557 (1990). While the specific process or processes involved in protein hydrolysis in muscle has not been identified, evidence is available linking the involvement of MCP in the accelerated breakdown of muscle proteins. See, for example, Furono, supra, and PCT Published Application WO 92/20804 (publication date: Nov. 26, 1992).
The levels of several proteins responsible for cell cycle regulation are controlled through ubiquitin-dependent degradation by the proteasome. Among these proteasome substrates are the tumor supressor protein p53 (Schneffer, M. et al. Cell 75:495-505 (1993)), the cyclin-dependent kinase inhibitor p27 (Pagano, M. et al., Science 269:682-685 (1995), and cyclin B (Glotzer, M. et al., Nature 349:132-138 (1991). Inappropriate degradation of these key regulatory proteins by ubiquitin-dependent proteolysis has been correlated with the development of tumors in the case of p53 and human papillomavirus-containing cervical carcinomas (Schneffer, M. et al., PNAS 88: 5523-5527 (1991); Hubbert, N. et al., J. Virol. 66:6237-6241 (1992)) and in the example of p27 in colorectal carcinomas (Loda, M. et al., Nature Med. 3:231-234 (1997)). Additionally, high levels of p27 are associated with a positive clinical outcome in colon carcinoma (Loda, supra; Fredersdorf. S. et al., PNAS 94:6380-6385)), breast carcinomas (Catzavalos, C. et al. Nature Med. 3:227-230 (1997); Porter, P. et al., Nature Med. 3:222-225 (1997); Fredersdorf, supra) and gastric carcinoma (Mori, M. et al. Nature Med. 3:593 (1997)). Treatment of transformed cells with proteasome inhibitors has been reported to result in accumulation of p27 (Drexler, H. C. A. PNAS 94:855-860 (1997)) and p53 (Shinohara, K. et al. Biochem J. 317:385-388 (1996); Lopes, U. et al. J. Biol. Chem. 272:12893-12896 (1997)) and to trigger apoptotic death of the cells. Therefore, compounds which inhibit the degradation of these growth inhibitory factors would be expected to cause cell cycle arrest and would be useful in the treatment of cancer and other proliferative diseases, including psoriasis and restenosis.
The transcription factor NF-.kappa.B stimulates expression of a wide variety of genes important in immune and inflammatory responses (Bauerle, P. and Henkel, T. Annu. Rev. Immunol. 12:141-179 (1994)). In unstimulated cells, NF-.kappa.B exists as an inactive cytoplasmic complex with an inhibitor protein, I.kappa.B. Upon stimulation of the cells, the I.kappa.B protein is ubiquitinated and degraded by the proteasome, activating NF-.kappa.B (Palombella, V. et al. Cell 78:773-785 (1994)). The resulting free NF-.kappa.B enters the cell nucleus where it initiates transcription.
Activation of NF-.kappa.B results in production of inflammatory cytokines such as tumor necrosis factor a, interleukin-2 and interleukin-6, and cell adhesion molecules including intracellular cell adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1-(VCAM-2), and E-selectin (Baeuerle and Henkel, supra). Suppression of the activation of NF-.kappa.B through inhibition of the proteasome would therefore provide a method for treatment of inflammatory conditions including arthritis, sepsis, and inflammatory bowel disease.
Activation of the cellular apoptotic program is a current strategy for treatment of human cancers. It has been demonstrated that X-irradiation and standard chemotherapeutic drugs kill some tumor cells through induction of apoptosis (Fisher, D. E. Cell 78, 539-542 (1994)). Unfortunately, the majority of human cancers at present are resistant to these therapies (Harrison, D. J. J. Patho. 175, 7-12(1995)). Activation of NF-.kappa.B has been demonstrated to render some tumor cells resistant to the pro-apoptotic effects of TNF-.alpha., cancer chemotherapeutic agents and radiation (Beg, A. And Baltimore, D. Science 274, 782-784(1996); Wang, C. Y. et al., Science 274, 784-787 (1996); Van Antwerp D., et al., Science 274, 787-789 (1996)). Administration of proteasome inhibitors to patients with cancer in combination with ionizing radiation or other chemotherapeutic drugs might enhance the efficacy of the pro-apoptotic agent.
Activated NF-.kappa.B is also required for replication of the human immunodeficiency virus (HIV) (Nabel, G. and Baltimore, D. Nature 326:711-713 (1987)). A process which inhibits activation of NF-.kappa.B could therefore be therapeutically beneficial to patients infected with HIV.
Cytosolic antigens are processed through ubiquitination and proteasome-catalyzed cleavage into peptides which are transported to the endoplasmic reticulum and bound to the MHC-1 complex. Proteasome inhibitors prevent MHC-1 antigen presentation without effect on MHC-II antigen processing (Rock, K. et al. Cell 78:761-771 (1994); Harding, C. et al. J. Immunol. 155:1767-1775 (1995)). Such inhibitors should therefore be useful in the treatment of diseases resulting from inappropriate antigen presentation, including autoimmune diseases and rejection of transplants.
Proteasome activity also is required in the life cycle of many parasitic organisms. For example, both the bloodstream and insect forms of the protozoan parasite Trypanasoma brucei encode a 20S proteasome which is believed to be required for the organism's survival (Hua et al. Mol. Biochem. Parasitol. 78, 33-46(1996); Lomo et al. Immunopharmacology 36, 285-293(1997); To and Wang FEBS Lett. 404, 253-262(1997)). Inhibitors of the proteasome have been demonstrated to prevent the transformation of Trypanosoma cruzi trypomastigotes into amastigotes, and the intracellular development of amastigotes into trypomastigotes (Gonzalez et al. J. Exp. Med. 184, 1909-1918(1996)). Proteasome inhibitors should therefore have utility in the treatment of diseases resulting from parasitic infections including, but not limited to, African sleeping sickness and malaria.
MCP activity has been implicated in several disease states. For example, abnormally high expression of MCP in human leukemic cell lines has been reported. Kumatori, A. et al. PNAS 87:7071-7075 (1990). Autoantibodies against MCP in patients with systemic-lupus erythematosus ("SLE") have also been reported. Arribas, J. et al. J. Exp. Med. 173:423-427 (1990).
Agents which are capable of inhibiting the MCP complex are needed; such agents would provide a valuable tool for both those conducting research in the area of, for example, MCP activity, as well as those in the medical fields in order to, for example, control the deleterious effects of abnormal or aberrant MCP activity. The present invention is directed to these important ends.